【作者】 季静娟；

【导师】 陈子江；

【作者基本信息】 山东大学 ， 临床医学（专业学位）， 2013， 博士

【摘要】 第一部分体外受精-胚胎移植周期中获卵数与妊娠结局之间的关系研究目的：已有的研究表明体外受精-胚胎移植(in vitro fertilization-embryo transfer, IVF-ET)治疗周期中获卵数和IVF结局相关,但首次IVF周期获卵数和鲜胚及累积活产率之间的关系尚不清楚。本研究首次探讨获卵数对首次IVF周期活产率的影响。方法：回顾性分析2007年4月～2011年12月于安徽省立医院生殖中心行IVF-ET助孕治疗的不孕症患者2455例。患者年龄为18-34岁；所有患者月经周期正常；均以标准黄体期长方案促排卵行第一次IVF周期助孕；未进行种植前遗传学诊断或筛查；非供卵、部分供卵、接受供卵或解冻卵IVF周期；非自然周期或还有剩余冷冻胚胎冻存IVF周期。超促排卵、体外授精-胚胎移植、黄体支持、胚胎冻融及冻胚移植等按照我中心常规进行。单因素分析确定对活产率有影响的预测因子。按获卵数将患者分为5组：A组(<5个),B组(6～10个),C组(11～15个),D组(≥16个)。比较各组受精率、可移植胚胎数、每起始周期鲜胚活产率、全胚冷冻率、每移植周期鲜胚活产率、每起始周期累积活产率、流产率及中重度卵巢过度刺激综合症(ovarian hyperstimulation syndrome, OHSS)发生率。多变量Logistic回归排除了其他对活产率有影响的预测因子后分析获卵数对鲜胚活产率及累积活产率的影响。结果：2455个周期中1333个周期获得活产,活产率54.30%。获得活产的患者较未活产患者年轻(P=0.001)。获得活产的患者每日促性腺激素(gonadotropin, Gn)用量及Gn总用量小于未活产患者(P<0.001,P<0.05)；但是,获得活产患者的Gn使用天数明显长于未活产患者(P<0.001)。二者之间不孕年限、受精方式、不孕类型、助孕指征差异无显著性。获得活产患者获卵数、受精率、可移植胚胎数明显高于未活产患者(P<0.001,)；相反,获得活产患者平均移植鲜胚数量明显少于未活产患者(P<0.05)。各获卵数组之间的比较显示,年龄和获卵数呈负相关(P<0.001)。随获卵数的增加,受精率下降(P<0.001),但可移植胚胎数却增加(P<0.001)。343个周期未移植新鲜胚胎,原因为：未获卵(9周期[0.37%]),受精障碍(26周期[1.06%]),无可移植胚胎(17周期[0.69%]),因OHSS或其他原因行全胚冷冻(290周期[11.81%])。随获卵数的增加,OHSS倾向取消鲜胚移植行全胚冷冻的患者增加(P<0.01)。每起始周期新鲜胚胎移植后活产率B、C两组显著高于A、D两组(P<0.01),D组鲜胚活产率下降可能与OHSS倾向取消鲜胚移植行全胚冷冻的患者增多有关。而每移植周期的鲜胚活产率和每起始周期的累积活产率随获卵数的增加而增加(P<0.01,0.005),分别从A组的38.69%和35.00%上升至D组49.07%和67.10%。同样,中重度OHSS的发生率随获卵数的增加而增加(P<0.01),各组流产率相比差异无显著性(P>0.05)。多变量Logistic回归分析以A组作为参照组,结果表明,对于每起始周期新鲜胚胎活产率,当排除年龄、超促排卵天数、Gn用药总量、受精率的影响后,修正ORs从B组的1.823(1.395-2.381)升高至C组的2.142(1.609-2.851)。但是D组修正ORs下降,为1.918(1.376-2.672)。超促排卵天数、Gn用药总量、受精率与每起始周期鲜胚活产率相关,但是年龄与每起始周期鲜胚活产率相关性不显著。对于每起始周期累积活产率,当排除年龄、超促排卵天数、Gn用药总量、受精率的影响后,修正ORs在B组和D组分别为2.232(1.708-2.918)、5.805(4.125-8.170),随获卵数增加而上升。超促排卵天数和受精率与累积活产率相关。结论：首次IVF周期中获卵数和鲜胚及累积活产率之间有很强的相关性。对年轻患者来说,卵巢高反应并不降低每起始周期IVF累积活产率,但是在IVF新鲜胚胎移植周期,卵巢高反应意味着因OHSS倾向取消鲜胚移植行全胚冷冻的患者增加。在首次IVF周期中,获得最佳活产率的获卵数是6-15个。第二部分TCRαβ+CD3+CD4-CD8-双阴性调节性T细胞在不明原因复发性流产中的作用及其机制研究目的：探讨TCRαβ+CD3+CD4-CD8-双阴性调节性T细胞(double negative regulatory T cells, DN Treg)在正常妊娠和不明原因复发性流产(unexplained recurrent spontaneous abortion, URSA)患者外周血和母胎界面的含量和功能的变化,了解DN Treg细胞在URSA中的可能作用。同时检测URSA患者主动免疫治疗前后外周血DN Treg细胞水平变化,探讨主动免疫治疗的可能机制。研究结果有助于阐明DN Treg细胞在诱导母胎免疫耐受及URSA中的作用,以期为URSA的治疗寻找新靶点。方法：①采集正常非孕妇女、正常早孕妇女、URSA患者以及接受主动免疫治疗前后的URSA患者外周血,用密度梯度离心法分离外周血单个核细胞,分别检测各组DN Treg细胞含量的变化。②采集URSA患者或正常早孕行人工流产妇女蜕膜组织,分别检测各组中DN Treg细胞含量。③提取外周血单个核细胞、蜕膜局部白细胞,分离纯化DN Treg细胞。用抗CD3/CD28包被磁珠体外诱发活化DN Treg细胞。④采集各研究对象的外周血,分离淋巴细胞并制成悬液,CFSE染色后加或不加自体活化后的DN Treg细胞,同时加入抗CD3/CD28磁珠,共刺激培养5天后,上流式细胞仪检测,CELL Quest软件获取并分析,计算出增殖细胞的比例(CD4+或CD8+T细胞中CFSE dim细胞的比例)。结果：正常早孕妇女蜕膜组织中DN Treg细胞占CD3+T细胞的比例均明显高于外周血,分别为5.26%±3.58%和1.56±1.07%,差异有显著性(P<0.01)。同样,URSA患者蜕膜组织中DN Treg细胞比例也明显高于外周血,分别为1.82%±0.85%和0.92%±0.68%(P<0.01)。正常妊娠和正常非孕妇女外周血DNTreg细胞比例分别为：1.56%±1.07%和1.21%±0.63%,差异无统计学意义(P=0.217)；而URSA患者外周血中DN Treg细胞的比例显著低于正常妊娠妇女外周血,分别为0.92%±0.68%和1.56%±1.07%(P=0.009)。同样,URSA患者蜕膜组织DN Treg细胞也含量明显低于正常妊娠妇女蜕膜组织,差异有统计学意义(P=0.011)。体外功能测定显示：URSA患者的外周血和蜕膜组织中的DN Treg细胞可明显抑制自体CD4+和CD8-T细胞的增殖。同样,正常早孕者蜕膜组织DN Treg细胞也可抑制自体CD4+和CD8+T细胞的增殖。二组之间蜕膜组织DN Treg细胞的抑制能力无明显差异。接受免疫治疗共有54名患者,治疗后至今已分娩17例,目前妊娠已达21-35周有8例,12-20周7例,15例患者妊娠后再次流产,7例患者未孕。将妊娠>12周及分娩定为治疗成功计算,治疗成功率为59.2%。主动免疫治疗后,URSA患者外周血DN Treg细胞比例较治疗前上升,但差异无显著性(P=0.151)。但32例妊娠成功者主动免疫治疗后外周血DNTreg细胞比例较治疗前显著升高(P<0.05),而15例妊娠失败者主动免疫治疗前后外周血DN Treg细胞比例无明显变化。结论：蜕膜是人DN Treg细胞在体内富集的部位之一。人DN Treg细胞在母胎免疫耐受中发挥重要的作用,外周血和蜕膜组织中]DN Treg细胞含量的减低可能与不明原因复发性流产的发生相关。通过提高URSA患者外周血中DN Treg细胞含量,可能是主动免疫治疗发挥作用的机制之一。更多还原

【Abstract】 Part Ⅰ:The optimum number of oocytes in IVF treatmentObjective:It has been reported that there is a strong association between oocyte number and in vitro fertilization-embryo transfer (IVF-ET) outcome.However, the association between the number of eggs retrieved and the live birth rate (LBR) both per fresh cycle and cumulatively per stimulation cycle are not known yet. The purpose of this study is to explore the relationship between oocyte number and LBR following first IVF treatment cycles.Method:A retrospective cohort study was performed. All patients aged18-34years with normal menstrual cycles and were stimulated with a long protocol at the Reproductive Medicine Center of Anhui Provincial Hospital, P.R. China from April2007to December2011were identified and reviewed. In our study, the patients’ own oocytes were used, and fresh or frozen-thawed embryos were transferred without pre-implantation genetic screening for embryo aneuploidies. For the purpose of this study, cycles involving egg donation, egg sharing, egg cryopreservation and frozen egg thawing were excluded from the analysis. Patients who underwent natural cycles IVF/intra cytoplasmic sperm injection (ICSI) or who had not become pregnant but still had frozen embryos left were also excluded from our study. All patients were undergoing their first IVF cycle. Thus,2455patients with one complete treatment cycle were included in this study.Univariate analysis was conducted to identify confounding factors that predict live birth outcome. Patients were categorized into four groups according to the number of oocytes retrieved:A(0-5), B (6-10), C (10-15), D (≥16)oocytes. Important aspects of the treatment outcome including fertilization rate, embryo transferable, fresh cycle LBR per started cycle and per transfer cycle, cumulative LBR, abortion rate and moderate-severe ovarian hyperstimulation syndrome (OHSS) rate were analyzed. Logistic regression analyses were conducted to identify independent correlates between each possible confounding factor especially oocyte number and live birth outcome after adjusting for other confounders that were identified in our univariate analysis.Result:2455FVF/ICSI long protocol cycles from our unit were analyzed in this study, and1333cycles (54.30%) result in live births. Women who achieved live births were significantly younger (P=0.001) and required a smaller amount of daily and total gonadotropins (P<0.001, P<0.05). However, the duration of ovarian stimulation in live birth patients was significantly longer than their no live birth counterparts (P<0.001). There were no significant differences in the duration of infertility, insemination method, type of infertility, diagnosis of infertility between live birth and non-live birth cycles. The number of oocytes retrieved, fertilization rate and embryos transferable were significantly higher in live birth cycles (P<0.001). On the contrary, the average number of fresh embryos transferred in live birth patients was significantly fewer than non-live birth patients (P<0.05).When divided by oocyte number, there was a negative association between age and oocytes yield (P<0.001). The fertilization rate decreased when more oocytes were retrieved (P<0.001).However, the embryos transferable increased with oocytes number (P<0.001). In343cycles (13.97%), fresh embryo transfer was not done for the following reasons:no oocytes retrieved (9cycles [0.37%]), failed fertilization (26cycles [1.06%]), no embryo transferable (17cycles [0.69%]), and all embryos cryopreservation because of the OHSS or other reasons (290cycles [11.81%]).The whole embryo cryopreservation rate for avoiding moderate-severe OHSS increase with oocyte number (P<0.01). The fresh cycle live birth per started cycle increased with oocytes number up to group B-C (6-15oocytes group) and then decreased (P<0.01) because of the highly whole embryo cryopreservation rate for avoiding moderate-severe OHSS. The fresh cycle LBR per transfer cycle and cumulative LBR per started cycle increased with oocyte number, from38.69%and35.00%of group A to49.07%and67.10%of group D (P<0.01,0.005). Also, the incidence of moderate-severe OHSS increased with the oocyte number (P<0.01). There was no significant difference in the abortion rate among the patient groups (P>0.05).In the logistic regression analyses:the0-5oocyte category was used as a reference. After adjustment for age, duration of stimulation, total dose of gonadotrophins and fertilization rate, the adjusted ORs for fresh embryo live birth per started cycle increased from1.823(1.395-2.381) in the6-10oocyte category to2.142(1.609-2.851) in the11-15oocyte category. However, the adjusted ORs of fresh embryo live birth decreased in the≥6oocyte category, being1.918(1.376-2.672). The duration of stimulation, total dose of gonadotrophins and fertilization rate had a significant effect on fresh cycle LBR. But for age, the effect was not significant. For the cumulative live births per started cycle, when adjusted for age, the duration of stimulation, total dose of gonadotrophins and fertilization rate, the adjusted ORs for cumulative live birth were2.232(1.708-2.918) in the6-10oocyte category,3.323(2.492-4.432) in the11-15oocyte category and5.805(4.125-8.170) in≥16oocyte category. Thus, the odds of cumulative live birth appeared to significantly increase with the ovarian response. The duration of stimulation and fertilization rate also had a significant effect on the cumulative LBR.Conclusion:There is a strong relationship between the number of oocytes and fresh as well as cumulative LBR following first IVF treatment. For young women, high ovarian response did not compromise IVF outcome. We anticipate these studies may lead to the development of more efficacious ovarian stimulation therapies for IVF. However in the initial fresh IVF cycle, a high ovarian response was associated with a high rate of cycle cancellation due to the risk of OHSS. The optimal number of oocytes for achieving the best chance of live birth in the first IVF cycle, and even higher chances of live birth in cumulative cycles, is somewhere between6and15oocytes. Part Ⅱ:Decidual and peripheral blood TCRαβ+CD3+CD4-CD8-double negative regulatory T cells in early pregnancy subjects and unexplained recurrent spontaneous abortion patientsObjective:To investigate the proportional and function changes of TCRαβ+CD3+CD4-CD8-double negative regulatory T cells (DN Tregs) in peripheral blood and maternal-fetal interface in unexplained recurrent spontaneous abortion (URSA) and normal pregnant (NP) women, and explore the probably role of human DN Tregs in the occurance of URSA. Also, the proportional changes of these cells in peripheral blood after lymphocyte therapy in URSA patients were investigated to find the rationale for this treatment. The results of this study is to explore the probable role that DN Tregs may play in the induction of maternal-fetal immunological tolerance and to find a new treatment target for URSA.Method:Heparinized venous blood was obtained from non-pregnant healthy women and women in normal early pregnancy. Similarly, heparinized venous blood was also obtained from patients with unexplained recurrent spontaneous early abortion after diagnosis and before curettage and URSA patients received alloimmunization. treatment. Peripheral blood mononuclear cells (PBMCs) were separated from venous blood by Ficoll-Hypaque gradients. Decidual samples were obtained from patients with induced abortion, representing early pregnant deciduas, and from patients with spontaneous abortion, representing abortion samples. The decidual mononuclear cells (leukocytes) were purified by the Ficoll-Hypaque method after mechanical disruption and filtration through a32-μm nylon mesh. The proportion of DN Tregs in CD3+T cells of each group were determined by flow cytometry. DN Tregs were isolated from PBMC and decidual mononuclear cells via FACS separation. DN Tregs were activated by beads coated with anti-CD3and anti-CD28antibodies in vitro. Lymphocytes suspensions were isolated from venous blood of each patient.After stained with CFSE, lymphocytes were cocultured with anti-CD3/CD28-coated beads in the presence or absence of alloactivated DN Tregs for5days. Proliferation of cells was determined by flow cytometry. Data were analyzed with CELL Quest software and proliferation rates (the propotion of CFSEdim cells in CD4+or CD8+T cells) were calculated.Result:The propotion of TCR-αβ+CD3+CD4-CD8-DN Tregs in the CD3+cell population were compared between decidual lymphocytes and peripheral blood lymphocytes. The proportions of DN Tregs in decidua were significantly higher than that in peripheral blood both in URSA patients (1.82%±0.85%vs0.92%±0.68%, P <0.01) and normal early pregnant women (5.26%±3.58%vs1.56±1.07%, P<0.01). In peripheral blood lymphocytes, the proportion of DN Tregs did not differ significantly between normal early pregnant and non-pregnant women (1.56%±1.07%vs1.21%±0.63%, P=0.217). However, proportion of peripheral blood αβTCR+CD3+CD4-CD8-T (DN Treg) cells of URSA patients differ significantly from non-pregnant women (0.92%±0.68%vsl.56%±1.07%, P=0.009). Also, proportion of DN Tregs in deicidal were significantly lower in URSA patients than normal early pregnant women (1.82±0.85%vs5.26±3.58%, P=0.011). Result of in vitro suppression assays show that for URSA patients, DN Tregs in peripheral blood lymphocytes and decidua have a suppressive effect on allo-CD4+and allo-CD8+T cell proliferation. Also, DN Tregs in decidua of normal early pregnant women have a suppressive effect on allo-CD4+and allo-CD8+T cell proliferation. The suppression effect of DN Tregs in decidua between the two groups did not differ significantly.54URSA patients had received immunotherapy.32patients achieved a successful pregnancy with17patients already delieverd,8patients pregnant for21-35week,7patients pregnant for12～20week.7patients had not achieving pregnancy.15patients reaborted after pregnancy. The successful rate after this therapy is59.2%. After allogeneic lymphocyte therapy, the proportion of DN Tregs increase, but the difference was not significant (P=0.151). However, for32patients achieving a successful pregnancy, the percentage of DN Tregs in peripheral blood had been enhanced after allogeneic lymphocyte therapy (P<0.05). While for the15patients reaborted after lymphocyte therapy, the proportion of DN Tregs in peripheral blood did not differed significantly.Conclusion:Decidua may be one of the favorite tissue localization of DN Tregs in human in case of pregnancy. Human DN Tregs may play an important role in the induction of maternal-fetal immunological tolerance, the propotion decrease of DN Tregs in peripheral blood lymphocytes and decidua may be associated with the occurrance of URSA. Enhancement of the percentage of DN Tregs in peripheral blood may be one of the rationales for this treatment.更多还原